title: Bi-PROF: bisulfite profiling of target regions using 454 GS FLX Titanium technology.
abstract: The use of next generation sequencing has expanded our view on whole mammalian methylome patterns. In particular, it provides a genome-wide insight of local DNA methylation diversity at single nucleotide level and enables the examination of single chromosome sequence sections at a sufficient statistical power. We describe a bisulfite-based sequence profiling pipeline, Bi-PROF, which is based on the 454 GS-FLX Titanium technology that allows to obtain up to one million sequence stretches at single base pair resolution without laborious subcloning. To illustrate the performance of the experimental workflow connected to a bioinformatics program pipeline (BiQ Analyzer HT) we present a test analysis set of 68 different epigenetic marker regions (amplicons) in five individual patient-derived xenograft tissue samples of colorectal cancer and one healthy colon epithelium sample as a control. After the 454 GS-FLX Titanium run, sequence read processing and sample decoding, the obtained alignments are quality controlled and statistically evaluated. Comprehensive methylation pattern interpretation (profiling) assessed by analyzing 10 (2)-10 (4) sequence reads per amplicon allows an unprecedented deep view on pattern formation and methylation marker heterogeneity in tissues concerned by complex diseases like cancer.
authors.affiliation: Department of Genetics/Epigenetics, FR8.3 Life Sciences, Saarland University, Saarbrücken, Germany.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Gries
authors.forename: Dirk
authors.initials: D
authors.lastname: Schumacher
authors.forename: Julia
authors.initials: J
authors.lastname: Arand
authors.forename: Pavlo
authors.initials: P
authors.lastname: Lutsik
authors.forename: Maria Rivera
authors.initials: MR
authors.lastname: Markelova
authors.forename: Iduna
authors.initials: I
authors.lastname: Fichtner
authors.forename: Jörn
authors.initials: J
authors.lastname: Walter
authors.forename: Christine
authors.initials: C
authors.lastname: Sers
authors.forename: Sascha
authors.initials: S
authors.lastname: Tierling
mesh_terms: D000818:Animals; D001483:Base Sequence; D015179:Colorectal Neoplasms / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics; D017931:DNA Primers / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000378:metabolism*; D014025:Titanium / Q000378:metabolism*; D017186:Titrimetry; D023041:Xenograft Model Antitumor Assays
chemical_list: D017931:DNA Primers; D013447:Sulfites; D014025:Titanium; C042345:hydrogen sulfite
keywords: BiQ Analyzer HT; DNA methylation; bisulfite profiling; colorectal cancer; next-generation sequencing